Venom composition and pain-causing toxins of the Australian great carpenter bee Xylocopa aruana

Most species of bee are capable of delivering a defensive sting which is often painful. A solitary lifestyle is the ancestral state of bees and most extant species are solitary, but information on bee venoms comes predominantly from studies on eusocial species. In this study we investigated the venom composition of the Australian great carpenter bee, Xylocopa aruana Ritsema, 1876. We show that the venom is relatively simple, composed mainly of one small amphipathic peptide (XYTX1-Xa1a), with lesser amounts of an apamin homologue (XYTX2-Xa2a) and a venom phospholipase-A2 (PLA2). XYTX1-Xa1a is homologous to, and shares a similar mode-of-action to melittin and the bombilitins, the major components of the venoms of the eusocial Apis mellifera (Western honeybee) and Bombus spp. (bumblebee), respectively. XYTX1-Xa1a and melittin directly activate mammalian sensory neurons and cause spontaneous pain behaviours in vivo, effects which are potentiated in the presence of venom PLA2. The apamin-like peptide XYTX2-Xa2a was a relatively weak blocker of small conductance calcium-activated potassium (KCa) channels and, like A. mellifera apamin and mast cell-degranulating peptide, did not contribute to pain behaviours in mice. While the composition and mode-of-action of the venom of X. aruana are similar to that of A. mellifera, the greater potency, on mammalian sensory neurons, of the major pain-causing component in A. mellifera venom may represent an adaptation to the distinct defensive pressures on eusocial Apidae.


Results
The venom of Xylocopa aruana is simple in composition and similar to that of Apis mellifera. We used a combined transcriptomic and mass spectrometry (MS)-based strategy to generate a full profile of the composition of polypeptides in venom from an individual adult female X. aruana (Fig. 1a). RNA extracted from the venom glands was used to generate a venom gland transcriptome. We obtained 24,904,864 demultiplexed paired-end reads from Illumina NextSeq RNA sequencing, which, following adaptor trimming, quality trimming and filtering and error correction, were assembled de novo using Trinity to yield a total of 43,374 contigs. Venom was collected by squeezing of the contents of the venom reservoir and venom duct into water. Liquid chromatography-tandem MS (LC-MS/MS) data from three venom samples (native; reduced and alkylated; reduced, alkylated, and trypsin-digested) were searched against a database comprising the translated venom gland transcriptome.
Analysis of the venom of X. aruana by LC-MS indicated that it was relatively simple (Fig. 1b). By topdown sequencing of the native and reduced and alkylated venom samples we identified two peptides ( Fig. 1b- Table 1). A 17 amino acid, cysteine-free peptide with an amidated C-terminus, which we called XYTX 1 -Xa1a, dominated the venom. The total ion chromatogram of the native venom, shown in Fig. 1b, illustrates the relative abundance of this peptide. Numerous derivatives (e.g. truncated version of the peptide) were also detected, although at much lower abundance, and are labelled with asterisks in Fig. 1b. We cannot confirm whether these derivatives are present in the natural venom or are an artefact of our venom collection technique. In the venom gland transcriptome two near-identical transcripts (probably representing either allelic variants or paralogues) encoded the mature peptide XYTX 1 -Xa1a, differing only by synonymous substitutions at two sites. Together, these accounted for 93.4% of venom component expression (Fig. 1e,f). The second peptide was 23 amino acids in length with four cysteine residues and an amidated C-terminus. A peak with mass [M + 4H] 4+ = 629.061 (theoretical [M + 4H] 4+ = 629.063) and MS/MS spectra corresponding to the monomeric peptide was detected in the native venom sample. No peaks with a mass corresponding to that of the dimeric peptide were detected, indicating that this peptide exists in the venom as a monomer and not as a dimer. This peptide, which we called XYTX 2 -Xa2a, accounted for 3.0% of venom component expression. Analysis of the native venom sample by matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) MS was consistent with the data obtained by LC-ESI-MS i.e. Four major peaks corresponded to XYTX 1 -Xa1a (and two derivatives) and XYTX 2 -Xa2a (Fig. S2).
Several other proteins were detected by bottom-up sequencing of the reduced, alkylated and trypsin-digested venom sample. Of these, a PLA 2 was the most highly expressed (3.4% of venom component expression) (Fig. 1c). This was almost identical (96% amino acid identity) to that reported in the venom of X. appendiculata (Uniprot: I7GQA7) and similar to those reported from Bombus and Apis venoms (Fig. S1). The remaining proteins were expressed at much lower levels, together constituting only 0.2% of venom component expression (Fig. 1c, Dataset 1). These proteins include some which have been implicated as toxins e.g. hyaluronidase, others likely serve a role in the production and/or maturation of the peptide toxins e.g. dipeptidyl peptidase-4 (DPP-4), while others  The high proportion of venom gland-derived reads encoding the two peptides XYTX 1 -Xa1a and XYTX 2 -Xa2a and the venom PLA 2 , as well as the assignment of the major peaks of the total ion chromatogram of the native venom, strongly suggested that together these three polypeptides represent the major components of X. aruana venom (Fig. 1).
Pharmacological activity of X. aruana venom peptides. We prepared XYTX 1 -Xa1a and XYTX 2 -Xa2a by solid phase peptide synthesis (SPPS). Oxidative folding of the linear XYTX 2 -Xa2a produced a single major peak which eluted at the same retention time as the native peptide in the venom (Fig. S3).
The mature peptide of XYTX 1 -Xa1a shares a similar primary structure with Xac-1, Xac-2 and melectin. Previous studies of these peptides have indicated amphipathic α-helical structure in membrane-mimicking solvents, degranulation of mast cells, and antimicrobial activity, all of which are consistent with a mode-of-action involving disruption of cell membranes [11][12][13] . We hypothesised that XYTX 1 -Xa1a would share the same activity. We tested, by whole-cell patch-clamp electrophysiology, the capacity of XYTX 1 -Xa1a to directly induce leak currents in cell membranes. For these experiments we used HEK293AD cells, which lack appreciable expression of the ion channels found in neurons. At 4 min after application of XYTX 1 -Xa1a (30 μM) we recorded leak currents at test potentials ranging from − 60 to + 60 mV in 10-mV increments from a holding potential of 0 mV every 6 s. At + 60 mV, we recorded currents of 3.3 ± 0.6 nA (mean ± SEM, n = 6 cells) compared with 0.08 ± 0.02 nA (n = 5 cells) for time-matched negative controls (application of extracellular solution (ECS)) ( Fig. 3a-c). These data are consistent with a membrane disrupting mode of action for XYTX 1 -Xa1a, similar to that of melittin (Fig. S4). XYTX 2 -Xa2a is similar in sequence to Bombus and Apis MCD-peptides and apamin. Apamin is a blocker of the mammalian small conductance calcium-activated potassium (K Ca , SK) channel K Ca 2.2 20 , while Apis MCD peptide is a blocker of Shaker-like voltage-gated potassium (K V ) channels K V 1.1 and K V 1.2 21 . We hypothesised that XYTX 2 -Xa2a might share similar activity. Thus, we tested XYTX 2 -Xa2a for activity on human K V 1.1, K V 1.2,    Fig. 5a-b,f), while application of XYTX 2 -Xa2a or venom PLA 2 had no direct effect on intracellular calcium levels ( Fig. 5d-f). We measured the potency of XYTX 1 -Xa1a in F11 cells (a mouse neuroblastoma × rat DRG cell line) where the peptide caused an increase of [Ca 2+ ] i with a median effective concentration (EC 50 ) of 5.2 ± 0.7 µM (n = 6) (Fig. 5c). In this assay, melittin was more potent (P = 0.0002, unpaired t-test; n = 6) with an EC 50 of 1.2 ± 0.1 µM (n = 6) (Fig. 5c).
Previous studies have shown that A. mellifera venom PLA 2 potentiates the haemolytic activity of melittin 22 , and more recently it was shown that the PLA 2 toxins of spitting cobra venoms potentiate the nociceptive effects the cobra venom cytotoxins 23 . We hypothesized that the nociceptive effects of XYTX 1 -Xa1a might also be potentiated by PLA 2 . Indeed, activation of DRG neurons by XYTX 1 -Xa1a was increased in the presence of venom PLA 2 (1 µM) to 93.2 ± 6.5% (P = 0.0036, versus XYTX 1 -Xa1a alone, unpaired t-test), which was accompanied by increased cell lysis (Fig. 5d-f). Cell lysis is illustrated in Fig. 5d,e by leakage of dye into the extracellular media. Activation of DRG neurons by XYTX 1 -Xa1a was not increased by the presence of XYTX 2 -Xa2a (45.8 ± 5.5% neurons; P = 0.4096, versus XYTX 1 -Xa1a-treated, unpaired t-test) (Fig. 5f).
These data suggest that XYTX 2 -Xa2a, apamin and MCD-peptide do not contribute to spontaneous pain (in mammals) associated with envenomation by these bees. We therefore tested whether they might contribute to Representative whole-cell current traces were recorded for (a) hK V 1.1, (b) hK V 1.2, and (c) hK V 1.3 using the voltage protocols shown above the raw current traces every 15 s in the absence (black, control) and presence of 100 nM XYTX 2 -Xa2a (orange) and positive control (TEA + for hK V 1.1 and hK V 1.3, and charybdotoxin (ChTx) for hK V 1.2, blue). (d) hK Ca 2.1 and (e) hK Ca 2.2 currents were elicited with voltage ramps to + 50 mV from a holding potential of − 120 mV every 15 s in the absence (black, control) and presence of XYTX 2 -Xa2a at the indicated concentration (orange) or apamin as a positive control (blue). The currents were corrected for ohmic leakage and then drawn as a function of test potential (E m ). The horizonal dashed line shows the zero current level, the vertical dashed line indicates the expected reversal potential for K + (− 86.5 mV, based on the Nernst equation). (f) Low affinity, concentration-dependent block of hK Ca 2.2 channels by XYTX 2 -Xa2a. Whole-cell hK Ca 2.2 currents were recorded using voltage ramps as for (e). Remaining current fraction (RCF) was calculated as I/I 0 where I 0 is the peak current at + 50 mV in the absence and I is the peak current at + 50 mV in the presence of XYTX 2 -Xa2a at equilibrium block at concentrations of 0.1, 1, 5, and 10 μM (empty circles), respectively. Points on the linear dose-response curve represent the mean of 4-6 independent measurements. The line was drawn using linear least squares fit (see Methods for details). The reciprocal of the slope of the best fitted line yielded an IC 50 of 25.1 ± 3.5 μM. Data are mean ± SEM. www.nature.com/scientificreports/ longer-lasting pain responses e.g. allodynia, as has been reported for other hymenopteran venom peptides 26,27 .
Using an automated Von Frey apparatus, we measured paw-withdrawal threshold to a mechanical stimulus at 1 and 4 h following intraplantar injection of either XYTX 2 -Xa2a, apamin or MCD-peptide (injected; 2 pmol/ paw), where we observed no difference to negative control (saline) injection (Fig. 6d).

Discussion
In this study, we analysed the venom composition and function of the Australian great carpenter bee Xylocopa aruana. While the venom of the eusocial A. mellifera is among the most studied of all venoms, there have been few studies on the venoms of solitary Apidae. One probable reason for this is, due to their solitary lifestyle, a greater difficulty in acquiring multiple specimens and therefore sufficient venom and venom-producing tissue for analysis. However as demonstrated here, advances in the sensitivity of mass spectrometry and nucleotide sequencing have now made it possible to analyse the complete venom composition of an individual bee. Working with an individual rather than multiple specimens comes with both advantages and disadvantages: One advantage is that our data were not confounded by intra-specific genetic polymorphisms, which can interfere with transcriptome assembly and conclusions on venom complexity. But this could also be viewed as a potential limitation, i.e. that the venom composition of our specimen may not be an accurate representation of the species. To resolve this comprehensively would require the individual analysis of multiple specimens. However we note that studies of the congeneric X. appendiculata 13 and X. violacea 14 which used multiple individuals, present  Solitary and eusocial Apidae contend with distinct defensive selection pressures i.e. Xylocopa spp. sting solely in self-defence, while the eusocial A. mellifera stings in both self-defence and defence of its colony, including against large vertebrates. Defensive adaptations in the eusocial Apidae include alarm pheromones, increased aggression and sting autotomy (in Apis), which likely serve to increase the dose of venom that can be delivered to an aggressor. One might expect that the differing selection pressures between Apis and Xylocopa would also be reflected in differences in venom composition. However, our data suggest that X. aruana and A. mellifera share a similar venom composition and venom mode-of action. The sole difference we observed was in the potency of the major pain-causing components-melittin is approximately fivefold more potent than XYTX 1 -Xa1a at activating mammalian sensory neurons. We speculate that the greater potency of melittin, from the eusocial A. mellifera venom, over XYTX 1 -Xa1a may represent an additional adaptation in response to the distinct defensive selection pressures associated with the transition to eusociality. While this is consistent with a previous report of a correlation between venom lethal capacity and colony weight in stinging hymenopterans 19 , broader taxon sampling within the Apidae and in other bees, and comparative evaluation of venom potency, will be valuable in further testing this hypothesis.
In both X. aruana and A. mellifera, one amphipathic pore-forming peptide is the major venom component and the primary pain-causing agent. Venom PLA 2 increases the pain-causing effects of the amphipathic peptide. Such toxin synergy is widely believed to occur in venoms, yet very few examples have been documented todate. Other examples include the toxin "cabals" of cone snail venoms, where several toxins with complementary activity work together to achieve rapid paralysis of the prey 28 . Similarly PLA 2 in cobra venoms potentiates the www.nature.com/scientificreports/ pain-causing activity of venom cytotoxins 23 . The potentiation, by venom PLA 2 , of the pain-causing effects of melittin and the melittin-like peptide XYTX 1 -Xa1a, in the venoms of A. mellifera and X. aruana, respectively, is a third example of toxin synergy. We found that bee venom PLA 2 also induced pain behaviours in its own right, and thus can also be considered a pain-causing agent. We did not resolve the mechanism by which this occurs, although it appears to be independent of direct activation of sensory neurons. In contrast to the other major venom components, the apamin-like peptides, which are blockers of potassium channels and make up the final major class of polypeptides in these venoms, did not cause spontaneous pain behaviours or allodynia and their contribution(s) in the context of defence and pain remains unclear. This study contributes to our understanding of the evolution, chemistry and pharmacology of the venoms of the Apidae.

Mass spectrometry. A combination of top-down proteomics of native and reduced and alkylated venom,
and bottom-up proteomics of reduced, alkylated and trypsin-digested venom was used to examine the venom composition of the individual X. aruana. Two aliquots of venom (10 μg each) were dried by vacuum centrifugation. Gas phase reduction and alkylation was performed according to the protocol described by Hale et al. 34 . 100 μL of reduction/alkylation reagent (50% (v/v) ammonium carbonate, 48.75% ACN, 1% 2-iodoethanol, 0.25% triethylphosphine was added to the lid of each 1.5 mL tube containing dried venom, which was then inverted, closed, and incubated at 37 °C for 90 min. One aliquot of reduced and alkylated venom was then digested by incubating with trypsin (20 ng/μL) overnight at 37 °C, according to the manufacturer's instructions (Sigma-Aldrich, St. Louis, MO, USA). Three venom samples (10 μg each)-native venom, reduced and alkylated venom, and reduced, alkylated and trypsin-digested venom-were analyzed by LC-MS/MS. Samples were separated on a Nexera uHPLC (Shimadzu, Kyoto, Japan) with a Zorbax stable-bond C18 column (2.1 × 100 mm; particle size, 1.8 μm; pore size, 300 Å; Agilent, Santa Clara, CA, USA), using a flow rate of 180 μL/min and a gradient of 1-40% solvent B (90% ACN and 0.1% formic acid (FA)) in 0.1% FA over 25 min, 40-80% solvent B over 4 min, and analyzed on an AB Sciex 5600 TripleTOF (SCIEX, Framingham, MA, USA; operated with Analyst TTF v1.8) mass spectrometer equipped with a Turbo-V source heated to 550 °C. MS survey scans were acquired at 300 to 1800 mass/charge ratio (m/z) over 250 ms, and the 20 most intense ions with a charge of + 2 to + 5 and an intensity of at least 120 counts were selected for MS/MS. The unit mass precursor ion inclusion window mass within 0.7 Da and isotopes within 2 Da were excluded from MS/MS, with scans acquired at 80 to 1400 m/z over 100 ms and optimized for high resolution. Using ProteinPilot v5.0 (SCIEX), MS/MS spectra were searched against the translated venom apparatus transcriptome (MS and MS/MS tolerance of 0.05 and 0.1 Da, respectively). False discovery rate analyses were generated by ProteinPilot default method, which uses a decoy database.
Transcripts encoding venom components were then manually examined using the Map-to-Reference tool of Geneious v10.2.6 35 , where two paralogues of XYTX 1 -Xa1a were reassembled. These were then reincorporated back into the complete transcriptome, estimation of transcript abundance repeated, and a second, final Pro-teinPilot search performed. Peptides identified by ProteinPilot were validated by comparison of experimentally derived MS/MS peaks against a theoretical peak list generated using MS-Product in ProteinProspector v5.22.1 (http:// prosp ector. ucsf. edu/ prosp ector/ cgi-bin/ msform. cgi? form= mspro duct).
LC-MS was used to compare the elution times of oxidised synthetic XYTX 2 -Xa2a and native XYTX 2 -Xa2a in the venom. 10 μg native venom was separated on a Nexera uHPLC with a Zorbax stable-bond C18 column, using a flow rate of 180 μL/min and a gradient of 1-40% solvent B (90% ACN and 0.05% TFA) over 18 min and analyzed on an AB Sciex 5600 TripleTOF mass spectrometer. 1 nmol oxidised synthetic XYTX 2 -Xa2a (red) was analysed under the same conditions. The elution time of the extracted ion chromatogram (XIC) of 629.0627 ± 0.05 m/z (theoretical (M + 4H) 4+ ion of XYTX 2 -Xa2a) was compared.
Melittin and bee venom PLA 2 were purchased from Sigma-Aldrich (St. Louis, MO, USA), and apamin and MCD-peptide were purchased from Alomone labs (Jerusalem, Israel).

Whole cell voltage-clamp electrophysiology. HEK293AD cells (American Type Culture Collection)
were cultured as previously described 36 . Cells were maintained on DMEM supplemented with 10% heat-inactivated FBS, 2 mM l-glutamine, pyridoxine and 110 mg/mL sodium pyruvate. Whole-cell patch-clamp experiments were performed using a QPatch 16X automated electrophysiology platform (Sophion Bioscience). The extracellular solution contained the following: 70 mM NaCl, 70 mM choline chloride, 4 mM KCl, 2 mM CaCl 2 , 1 mM MgCl 2 , 10 mM Hepes, and 10 mM glucose (pH 7.4 with NaOH; 305 mosmol). The intracellular solution contained the following: 140 mM CsF, 1 mM:5 mM EGTA/CsOH, 10 mM Hepes, and 10 mM NaCl (pH 7.3 with CsOH; 320 mosmol). From a holding potential of 0 mV each recorded cell was subjected to a series of 50-ms voltage pulses that ranged from − 60 to + 60 mV in 10-mV increments. Recordings were made prior to and 4 min after the addition of either ECS (negative control) or XYTX 1 -Xa1a (10 µM). Data are mean ± SEM of 5-6 experiments and fitted to a simple linear regression.
Chinese Hamster Ovarian (CHO) cells (American Type Culture Collection) were grown in DMEM-high glucose supplemented with 10% FBS, 2 mM l-glutamine, 100 U/mL penicillin-g, and 100 μg/mL streptomycin (Invitrogen) at 37 °C in a 5% CO 2 and 95% air humidified atmosphere. Cells were passaged twice per week following a 7-min incubation in PBS containing 0.2 g EDTA/L (Invitrogen). hK V 1.1, hK V 1.2, hK Ca 2.1, and hK Ca 2.2 channels were transiently expressed in CHO cells using Lipofectamine 2000 (Invitrogen Carlsbad, CA), following the manufacturer's protocol and were cultured under standard conditions. For recording hK V 1.1, hK V 1.2, and hK Ca 2.1 currents GFP-tagged ion channel vectors were used. The hK Ca 2.2 channel plasmid was transiently co-transfected with a plasmid encoding the green fluorescent protein (GFP) at a molar ratio of 1:10. Transfected cells were washed twice with 2 mL of ECS (see below) and replated onto 35-mm polystyrene cell culture dishes (Cellstar, Greiner Bio-One). Currents were recorded 24 to 48 h after transfection. GFP-positive transfectants were identified with a Nikon Eclipse TS100 fluorescence microscope (Nikon, Tokyo, Japan) using bandpass filters of 455-495 nm and 515-555 nm for excitation and emission, respectively and were used for current recordings (> 70% success rate for co-transfection). hK V 1.3 currents were recorded on activated lymphocytes 3 to 4 days after activation. The human veinous blood was obtained from anonymized healthy donors. The peripheral blood mononuclear cells were isolated by Histopaque1077 (Sigma-Aldrich Hungary, Budapest, Hungary) density gradient centrifugation. Cells obtained were resuspended in RPMI 1640 medium containing 10% fetal calf serum (FCS, Sigma-Aldrich), 100 μg/mL penicillin, 100 μg/mL streptomycin, and 2 mM l-glutamine, seeded in a 24-well culture plate at a density of 5-6 × 10 5 cells/mL, and grown in a 5% CO 2 incubator at 37 °C for 3-5 days. Phytohemagglutinin A (Sigma-Aldrich) was added in 10 μg/mL concentrations to the medium to amplify the K V 1.3 expression. Cells were washed gently twice with 2 mL of ECS (see below) for the patch-clamp experiments. Standard whole-cell patch-clamp method 37 was used to record ionic currents. Micropipettes were pulled in four stages by using a Flaming Brown automatic pipette puller (Sutter Instruments, San Rafael, CA) from Borosilicate Standard Wall with Filament aluminum-silicate glass (Harvard Apparatus Co., Holliston, MA) with tip diameters between 0.5 and 1 μm and heat polished to a tip resistance ranging typically 2-8 MΩ. All measurements were carried out by using Axopatch 200B amplifier connected to a personal computer using Axon Digidata 1550A data acquisition hardware, respectively (Molecular Devices Inc., Sunnyvale, CA). In general, the holding potential was − 120 mV. Records were discarded when leak at the holding potential was more than 10% of the peak current at the test potential. Experiments were done at room temperature ranging between 20 and 24 °C. Data were analysed using GraphPad Prism 8 (Graphpad, CA, USA) and pClamp10.5 software package (Molecular Devices Inc., Sunnyvale, CA). Before analysis, whole-cell current traces were corrected for ohmic leakage and were digitally filtered with a three-point boxcar smoothing filter. For hK Ca 2.1-2 the reversal potential for K + was determined and only those currents were analyzed for which the reversal potential fell into the range of the theoretical reversal potential ± 5 mV (− 86.5 ± 5 mV). For hK V  were dissolved in the ECS supplemented with 0.1 mg/mL BSA (Bovine Serum Albumin). Bath perfusion around the measured cell with different extracellular solutions was achieved using a gravity flow micro perfusion system at a rate of 0.5 mL/min. Excess fluid was removed continuously. For measurements of currents on hK V 1.1-3 voltage steps to + 50 mV were applied from a holding potential of − 120 mV every 15 s and the peak current was measured. hK Ca 2.1-2 currents were elicited every 15 s with voltage ramps to + 50 mV from a holding potential of − 120 mV. The remaining current fraction (RCF) at a given molar concentration was calculated as I/I 0 , where I 0 is the peak current at + 50 mV in the absence and I is the peak current at + 50 mV in the presence of XYTX 2 -Xa2a at equilibrium block at a given concentration, respectively.  (1 µM) or XYTX 2 -Xa2a (1 µM), then at 1 min with XYTX 1 -Xa1a (in assay solution ± venom PLA 2 (1 µM) or XYTX 2 -Xa2a (1 µM)) and monitored for 2 min before being replaced with assay solution and then KCl (30 mM; positive control). Experiments involving the use of mouse tissue were approved by the University of Queensland Animal Ethics Committee (UQ AEC; approval number TRI/IMB/093/17). F11 (mouse neuroblastoma × DRG neuron hybrid; European Collection of Authenticated Cell Cultures) were cultured as previously described 36 . Cells were maintained on Ham's F12 media supplemented with 10% FBS, 100 µM hypoxanthine, 0.4 µM aminopterin, and 16 µM thymidine (Hybri-Max, Sigma Aldrich). 384-well imaging plates (Corning, Lowell, MA, USA) were seeded 24 h prior to calcium imaging, resulting in ~ 90% confluence at the time of imaging. Cells were loaded for 30 min at 37 °C with Calcium 4 assay component A in physiological salt solution (PSS; 140 mM NaCl, 11.5 mM d-glucose, 5.9 mM KCl, 1.4 mM MgCl 2 , 1.2 mM NaH 2 PO 4 , 5 mM NaHCO 3 , 1.8 mM CaCl 2 , 10 mM HEPES) according to the manufacturer's instructions (Molecular Devices, Sunnyvale, CA). Ca 2+ responses were measured using a FLIPR TETRA fluorescent plate reader equipped with a CCD camera (Ex: 470 to 490 nm, Em: 515 to 575 nM) (Molecular Devices, Sunnyvale, CA). Signals were read every second for 10 s before, and 300 s after, the addition of peptide (in PSS supplemented with 0.1% BSA).

Pain behaviour experiments.
Male adult (6 weeks old) C57BL/6J mice were used for behavioral experiments. To facilitate injections mice were briefly anesthetized using 2.5% isoflurane. Each peptide diluted in saline containing 0.1% bovine serum albumin (BSA), was administered in a volume of 20 µL into the hind paw by shallow intraplantar injection. Negative control animals were injected with saline containing 0.1% BSA. Following injection, spontaneous pain behaviour events were counted from video recordings by a researcher blinded to the treatments. Mechanical paw withdrawal thresholds were measured 1 and 4 h following injection using automated Von Frey apparatus (MouseMet; Topcat Metrology).
For calcium imaging experiments of mouse DRG neurons and F11 cells, treatment groups were compared using unpaired t-tests. For analysis of spontaneous pain, sum of pain behaviour counts at 30 min of treatment groups were compared using one-way ANOVA with Tukey's multiple comparisons test. Statistical significance was defined as P < 0.05. All data are presented as mean ± SEM.

Data availability
Prepropeptide sequences of XYTX 1 -Xa1a, XYTX 2 -Xa2a and the X. aruana venom PLA 2 have been deposited with GenBank, under accessions: ON586842, ON586843 and ON5868424, respectively. RNA-seq reads have been deposited in the NCBI sequence read archive under accessions SRR22306546. The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE 38